简并寡核苷酸引物PCR在荧光原位杂交检测染色体微缺失综合征中的应用

摘要

Objective To optimize the work of probe preparation used for fluorescence in situ hybridization(FISH) and aplly FISH analysis for the detection of deletions 7q1 1.2,15q11.2,and 22q1 1.2.Methods The DNA of bacterial artificial chromosome (BAC) was amplified by degenerate oligonucleotide primed PCR (DOP-PCR).Indirect labeling of the amplified products was performed by nick translation.Subsequently,the prepared FISH probes were used for detection of chromosome microdeletion.Results By means of DOP-PCR,we got sufficient amounts of FISH probes which provided clear hybridization signals and low backgroud.FISH analysis showed one case with of 15 q 11.2 deletion and another case with a mosaic 7q1 1.2 deletion.Conclusion The optimized method developed by us enables generation of probes to run faster without spending much time on isolating BAC-DNA.The probes could be applied in FISH analysis for the detection of microdeletion syndromes.%目的 优化荧光原位杂交(fluorescence in situ hybridization,FISH)所需探针的制备工作,应用FISH检测染色体7q1 1.2、15q11.2以及22q1 1.2缺失.方法 采用简并寡核苷酸引物聚合酶链式反应(degenerate oligonucleotide primed PCR,DOP-PCR)的方法,扩增人工细菌染色体(bacterial artificial chromosome,BAC) DNA,通过切口平移对扩增的DNA进行间接标记,使用制备好的探针检测染色体微缺失.结果 DOP-PCR方法得到的BAC-DNA扩增产物,经标记后用于FISH,可以观察到清晰且背景低的信号;FISH结果确认了1例染色体7q11.2微缺失嵌合体、1例染色体15q11.2缺失.结论 经过优化的实验方法能够快速制备探针,节约抽提BAC-DNA所需的时间,可以应用于FISH以检测微缺失综合征.